The ability to incorporate specific genes into the genome of mammalian embryos has provided a useful in vivo system for the analysis of gene control and expression. The high efficiency transformation of cultured mammalian cells has been accomplished by direct microinjection of specific DNA sequences into the cell nucleus (Capecchi, M., Cell 22:479-488 (1980)). Gordon, J. W. et al. (Proc. Natl. Acad. Sci. USA 77:7380-7384 (1978)) demonstrated that DNA could be microinjected into mouse embryos, and found in the resultant offspring. Thus, at the present time, the ability to produce certain transgenic mice is well described and well known in the art.
The basic procedure used to produce transgenic mice requires the recovery of fertilized eggs from the oviducts of newly mated female mice. DNA which contains the gene desired to be transferred into the mouse is microinjected into the male pronucleus of each fertilized egg. Microinjected eggs are then implanted into the oviducts of one-day pseudopregnant foster mothers and carried to term (Wagner, T. E. et al., Proc. Natl. Acad. Sci. USA 78:6376-6380 (1981)). The newborn mice are then tested for the presence of the microinjected DNA by means known in the art and appropriate to detect the presence of the microinjected DNA.
Using essentially this procedure, Wagner, T. E. et al., successfully produced an adult mouse capable of expressing rabbit B-globin. This mouse was further capable of producing offspring which also expressed the rabbit B-globin gene. Additional cloned immunoglobulin genes have been introduced into mouse germ lines to produce transgenic mice which produce the cloned immunoglobulin. (Grosschedl, R. et al., Cell 38:647-658 (1984)), Brinster, R. L. et al., Nature 306: 332-336 (1983)). Palmiter, R. D. et al. (Nature, 300:611-615 (1982)) produced transgenic mice which carried the mouse metallothionein I promoter fused to the structural gene for rat growth hormone. These mice were found to produce elevated levels of rat growth hormone, and to exhibit dramatically enhanced growth. Brinster, R. L. et al., Cell, 37:367-379 (1984)) injected the SV40 early region genes and a metallothionein fusion gene into fertilized mouse eggs, using essentially the method described above. A high percentage of the transgenic mice which developed from these eggs developed tumors within the choroid plexus. SV40 T-antigen-mRNA and protein were readily detected in this affected tissue. However, SV40 T-antigen gene expression was barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depended upon the activation of the SV40 genes. This work indicated that it was possible to microinject the genes which encode for a tumor virus into a mouse embryo and thereby produce a mouse with a predisposition to oncogenesis. Swift, G. M. et al. (Cell, 38:639-646 (1984)) produced a transgenic mouse which selectively expressed the gene for rat pancreatic elastase I at high levels in the mouse pancreas.
Cloned genes can be transferred into the mouse germ line by microinjection into the pronuclei of mouse zygotes. Such microinjected genes frequently integrate into chromosomes, are retained throughout development and are transmitted to offspring as Mendelian traits. (Grosschedl, R. et al.; Wagner, T. E. et al.). Investigators have reported that transgenic mice may be produced at an efficiency of between 10 and 30 percent using this procedure. Microinjected foreign genes have shown a tendency to be expressed in transgenic mice (Brinster, R. L. et al., Swift, G. M. et al.). Such expression, however, cannot be presumed. Lacy, E. et al., Cell 34:343-358 (1983) disclose a transgenic mouse containing the human B-globin gene that generally does not express this gene. Similarly, the rat and human growth hormone genes have been found to not be expressed under the control of their own promoters in transgenic mice which carry these genes (Wagner, T. E. et al.; Hammer, C. et al.). Expression of the chicken transferrin gene has been found to be enhanced only moderately in the liver (where it normally is preferentially expressed), relative to other tissues in a transgenic mouse which contains the chicken transferrin gene (McKnight, G. S. et al., Cell 34:335-341 (1983)). Thus, although the procedure for producing transgenic mice has been described in the art, the ability to produce specific transgenic mice which properly express the cloned gene remains imprecisely defined. It has been theorized that these aberrant results reflect the possibility that the cloned DNA has integrated into an essential region of the mouse chromosome that modifies its expression, or has undergone mutation or rearrangement in the process of integrating into the mouse chromosome.
Upon entry into the nucleus of the unicellular fertilized egg, the cloned gene fragments are believed to integrate at random sites on the mouse chromosomes. Once the cloned DNA has integrated into these sites it is, in general, stable and leads to the heritable transmission of these genes into progeny mice. Thus, the extremely variable expression of thymidine kinase in transgenic mice which carried the herpes simplex virus (type 1) gene is believed to result from a genetic rearrangement that occurred when the thymidine kinase fusion gene integrated into the mouse chromosome (Palmiter, R. D. et al., Cell, 36:869-877, (1984)).
The general method for producing transgenic mice is described in Wagner, T. E. et al., European Patent Application No. 81,570 (corresponding U.S. patent application No. 273,239) which specifically discloses a mouse expressing rabbit beta-globin gene in its erythrocytes.
Efforts to introduce the gene for human insulin into mice have been reported previously (Illmensee, K. et al., "Nuclear and Gene Transplantation in the Mouse." In: Brown B. D., Fox C. F., Eds. Developmental Biology Using Purified Genes, Academic Press, New York, pp 607-629 (1981); Burki, K. et al., Embo J. 1:127-131 (1982); Van der Putten, H. et al., Mol. Gen. Genetic 198:128-138 (1984)). These efforts resulted in the production of a mouse which contained the human insulin hormone gene sequences. The transgenic mouse produced by these laboratories was, however, unable to transmit the human insulin gene sequences to its progeny. Van der Putten, H. et al., showed that although progeny mice derived from a transgenic (insulin sequence bearing) mouse retained DNA sequences which had flanked the insulin gene sequence region, the progeny mice lacked all human insulin sequences. To explain how the insulin bearing sequences could have been deleted from the transgenic mice, while the flanking regions had been retained in these mice, Van der Putten, H. et al., postulated a germ line-specific excision event which specifically removed the human insulin sequences. Van der Putten, H. et al., concluded that the excision of the human insulin gene sequences might be a general phenomenon occurring during an initial step in the formation of the germ line in the transgenic female. The procedure employed by Van der Putten, H. et al. used a high number of circular DNA molecules containing the human insulin gene for the microinjections.
In conclusion, the prior art teaches the ability to produce certain transgenic mice which are capable of a tissue specific expression of a cloned gene. The prior art, however, also teaches the difficulty of specifically producing a transgenic mouse which expresses the human insulin gene. Although it has been possible to produce transgenic mice which carry DNA sequences of the gene for human insulin, these mice are not known to produce human insulin and have not been able to produce offspring which either express or carry the human insulin gene (Van der Putten H. et al., Mol. Gen. Genetic 198:128-138 (1984)).
It is therefore of great interest to develop a method for producing a mouse capable of expressing a human hormone or analogue of a human hormone and which can heritably transmit the capacity to express this hormone to its progeny mice.